Oxidative modification of proteins is involved in the pathophysiological changes of many human disorders and aging. Because the pattern of protein modification by one form of reactive oxygen species (ROS) may differ from that of another, elucidation of the patterns elicited by various forms of ROS may facilitate the development of diagnostic tools for identification the ROS involved in a given disorder, and the development of therapeutic strategies for its treatment. The present study was undertaken to examine the pattern of protein modification elicited by alkylperoxy radical and their derivatives. To this end, we exposed glutamine synthetase and the polypeptide melittin to solutions containing 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH), which is known to decompose in aqueous aerobic solutions to yield alkylperoxy radicals, alkylperoxides and alkoxy radicals. Under our experimental conditions (physiological pH, 37 degrees centigrade), the AAPH-dependent formation of alkylperoxide increased linearly with time and led to 40% and complete inactivation of glutamine synthetase in 1 and 4 hours, respectively. Complete loss of activity was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyrosine residues, 5 of 16 methionine residues, and all (only 2) of the tryptophan residues per enzyme subunit. None of the other amino acid residues of the enzyme were modified. N-formyl kynurenine was identified as the major product of tryptophan oxidation in melittin. The pattern of protein oxidation by AAPH derived ROS is therefore similar to that previously shown to be caused by exposure to ozone.